1. Field of the Invention
The present invention relates to a process for preparing an antigen of Treponema (Treponema pallidum, hereinafter sometimes abbreviated to TP) which is used as a reagent for diagnosing syphilis. More particularly, the present invention relates to a method for preparing an antigen which enables to prepare a diagnostic agent for syphilis, exhibiting high specificity and being able to detect primary syphilis. Further, this invention relates to a diagnostic reagent for syphilis and a method for preparing the same.
2. Prior Arts
Diagnostic methods have been performed which utilize the antigen-antibody reaction of TP antigens and anti-treponemal antibodies (hereinafter abbreviated to TP antibody) in sera from syphilitic patients. Among such methods, TPHA (Treponema pallidum hemaggultination assay test) has been widely used in recent years because of the advantages in its sensitivity, specificity and convenience in operation. Therefore, the TPHA has been a typical diagnostic method for syphilis.
The antigen solution originated from TP and used in the above-mentioned method is prepared as follows: First, TP is inoculated and cultivated in rabbit testes. The treponemes are extracted and suspended in a suitable buffer and then disrupted by homogenizer, sonicator and so forth. Thus disrupted treponemes with or without solubilization was used as the antigen solution for sensitization.
However, the prior art has the following drawbacks. Specifically, primary syphilis can not sufficiently be detected by the diagnostic agent for syphilis made from the conventional TP antigen solution. In other words, the conventional TPHA test or the like does not show a positive result in most cases until 2 to 3 months after syphilitic infection. Accordingly, there is a great problem that, in order to accomplish reliable diagnosis for primary syphilis, a diagnostic reagent using a lipoidal antigen (cardiolipin) should be used together with the TPHA method. Although the reagent using lipoidal antigens is sensitive to primary syphilis, nonspecific reactions are often observed.
In a syphilitic antibody detection test such as TPHA, sensitivity of reagent to a primary antibody (Ig-M) is lower than an advanced antibody. This was caused by impurities in the antigen solution used for the reagent. Namely, in the conventional TPHA, the TP antigen solution used for sensitization of animal erythrocytes inevitably includes impurities due to the preparation method. 90% or more of impurities are proteins originated from rabbit testes in which TP is cultivated, or from TP components having no antigenicity. Consequently, a significant quantity of impurities is incorporated in the TP antigen solution, so that the primary antibody (Ig-M) cannot be detected.
The antigen solution used for the conventional TPHA inevitably includes components originated from rabbit tissue due to its preparation method, which cause nonspecific reaction. Therefore, in order to reduce the nonspecific reaction, some components originated from rabbit tissue were added to the buffer of TPHA for absorbing heterophil antibodies in serum to be tested.
In order to solve the above-mentioned problems, Japanese Unexamined Patent Application No. SHO 58(1983)-71457 discloses a technique using an antigen fraction which is obtained by removing fractions having specific gravity of 1.01 or less from the extracted treponemal suspension.
However, a significant quantity of impurities is still mixed in the antigen fraction, because the fractionation method by the difference of specific gravity does not provide a strict separation of the antigen fraction from rabbit tissue. The aforesaid Japanese Application states that the above method is applicable even after the disruption of treponemes. However, sodium diatrizoate or the like used for density gradient reagents is inevitably incorporated in the antigen solution in this method, and hence, a process for removing the density gradient reagents should be required. Accordingly, this method would not be applied after the disruption of treponemes.
An immunological diagnostic reagent is generally prepared by immobilizing an antigen or antibody on a hydrophobic carrier (e.g., plastic particles such as latex particles, cellulose powder, polystyrene, polypropylene or nylon particles; membrane of nitrocellulose or nylon; erythrocytes treated with tannic acid; or agarose gel). Known immobilization methods include a method by physical adsorption wherein an antigen or antibody is in contact with a hydrophobic carrier in an aqueous medium and a method wherein an antigen or antibody is covalently bonded to a carrier having an amino group or a carboxylic group on its surface. The former method utilizing physical adsorption is widely used in view of manufacturing efficiency, convenience and being easy to reproduce the product of the same quality.
In the case of immobilizing an antigen or antibody by physical adsorption, an approximately neutral buffer comprising a salt and a buffering agent is usually used as the aqueous medium. A surfactant is not employed as the aqueous medium, since the surfactant is considered to interfere with the immobilization of antigen or antibody on carrier. Specifically, the surfactant is considered to decrease the hydrophobic interaction in immobilization by physical adsorption.
The surfactant is also considered to interfere with the immobilization of TP antigens on carriers. In other words, the efficiency of immobilization is substantially decreased in the presence of the surfactant, with the result that it is difficult to prepare an excellent diagnostic reagent.